Compositions and methods for treatment of osteoporosis and other indications

ABSTRACT

The present invention generally relates to compositions and methods for treatment of subjects having or at risk of osteoporosis or other conditions. In some cases, the composition may include nitric oxide. The nitric oxide may be present within a first phase comprising a lecithin, such as phosphatidylcholine. In certain embodiments, the lecithin is present in liposomes, micelles, or other vesicles containing nitric oxide. The composition can take the form of a gel, a cream, a lotion, an ointment, a solution, a solid “stick,” etc., that can be rubbed or sprayed onto a suitable portion of the skin. Other aspects of the present invention are generally directed to methods of making or using such compositions, methods of promoting such compositions, kits including such compositions, or the like.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/623,004, filed Sep. 19, 2012, entitled “Compositions andMethods for Treatment of Osteoporosis and Other Indications,” byNicholas V. Perricone, incorporated herein by reference in its entirety.

FIELD

The present invention generally relates to compositions and methods fortransdermal drug delivery, including treatment of osteoporosis and otherconditions.

BACKGROUND

Osteoporosis and other bone destructive disorders are a major problem inour health system due to an aging population. Primary osteoporosistreatments focus on decreasing bone destruction by reducing theformation and maturation of osteoclasts. Osteoporosis treatments includeestrogen replacement therapy, administration of bisphosphonates,selective estrogen receptor modulators, calcitonin, and antibodies suchas denosumab. However, such therapies are sometimes associated withadverse effects, e.g., breast cancer, osteonecrosis of the jaw,hypercalcemia, and hypertension. Accordingly, there exists a need fornew treatments for osteoporosis.

SUMMARY

The present invention generally relates to compositions and methods fortransdermal drug delivery, including treatment of osteoporosis. Thesubject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

In one aspect, provided herein is a method comprising administering, tothe skin of a subject having or at risk of osteoporosis, a compositioncomprising an effective amount of nitric oxide and a carrier having aphosphatidylcholine component entrapping the nitric oxide.

In another aspect, provided herein is a method comprising contacting theskin of a subject having or at risk of osteoporosis, with a compositioncomprising an emulsion comprising a first phase comprising nitric oxideand lecithin, and a second phase comprising an emulsifier, wherein thelecithin is present at least about 0.25% by weight of the composition,and wherein the first phase comprises no more than about 250 ppm ofwater by weight of the composition.

Several methods are disclosed herein of administering a subject with acompound for prevention or treatment of a particular condition. It is tobe understood that in each such aspect of the invention, the inventionspecifically includes, also, the compound for use in the treatment orprevention of that particular condition, as well as use of the compoundfor the manufacture of a medicament for the treatment or prevention ofthat particular condition.

In another aspect, the present invention encompasses methods of makingone or more of the embodiments described herein, for example, acomposition comprising nitric oxide. In still another aspect, thepresent invention encompasses methods of using one or more of theembodiments described herein, for example, a composition comprisingnitric oxide.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention. In cases where the present specificationand a document incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows results from the laser Doppler assay in four human studyparticipants.

DETAILED DESCRIPTION

The present invention generally relates to compositions and methods fortreatment of subjects having or at risk of osteoporosis and otherconditions. In some cases, the composition may include nitric oxide. Thenitric oxide may be present within a first phase comprising a lecithin,such as phosphatidylcholine. In certain embodiments, the lecithin ispresent in liposomes, micelles, or other vesicles containing nitricoxide. The composition can take the form of a gel, a cream, a lotion, anointment, a solution, a solid “stick,” etc., that can be rubbed orsprayed onto a suitable portion of the skin. Other aspects of thepresent invention are generally directed to methods of making or usingsuch compositions, methods of promoting such compositions, kitsincluding such compositions, or the like.

According to one aspect of the present invention, a composition asdescribed herein is used to treat osteoporosis or other conditions. To“treat” a disorder means to reduce or eliminate a sign or symptom of thedisorder, to stabilize the disorder, and/or to reduce or slow furtherprogression of the disorder. Thus, in one set of embodiments, theapplication of nitric oxide, e.g., in a nitric oxide-containing matrix,to skin may result in clinical improvement. In some cases, the deliveryof nitric oxide to the skin, e.g., to the epidermis and/or dermis, maybe achieved at a controlled rate and/or concentration. Without wishingto be bound by any theory, it is believed that nitric oxide therapy isbeneficial in the treatment of osteoporosis due to the relationshipbetween the NO-cGMP pathway and bone health (see, e.g., Wimalawansa,Expert Opin. Pharmacother. 2008, 9:3025). Supplementation with nitricoxide in deficient individuals allows for biological homeostasis.

In some embodiments, the nitric oxide in the composition is stable atroom temperature, and may remain active for extended periods of time,e.g., at least 1 year, at least 1.5 years, at least 2 years, at least2.5 years, at least 3 years, at least 4 years, etc. The nitric oxide maybe released, for example, when the composition is exposed to an aqueousenvironment, e.g., within the body. Without wishing to be bound by anytheory, it is believed that when the composition is applied to the skin,the liquid crystal structure collapses, delivering nitric oxide to adesired area of treatment. The concentration of the nitric oxide insidethe liquid crystal matrix can be varied in terms of concentration. Thematrix also may act as a sustained release and delivery system in someembodiments. It is also believed that the liquid crystal is highlypenetrating, such that nitric oxide can be delivered to the epidermis,dermis and dermal vascular for systemic release as well as tosubcutaneous fat, at least under some conditions.

Thus, in one set of embodiments, application of nitric oxide, e.g., in apenetrating matrix gel delivering the active nitric oxide, may act as apreventative of and/or be used to treat osteoporosis. In another set ofembodiments, a composition as described herein is applied to the skin ofa subject, e.g., one having or at risk of osteoporosis.

In some cases, the subject may be one that already has osteoporosis.However, in other cases, the subject may not necessarily haveosteoporosis, but may be one that is at risk of developing osteoporosis.For instance, the subject may be one that is elderly or soon to beelderly. In some cases, the subject may be greater than 40, greater than45, greater than 50, greater than 55, greater than 60, greater than 65,greater than 70, greater than 75, greater than 80, or greater than 85years old. The subject may a post-menopausal woman. The subject may alsobe one with a family history of osteoporosis. The subject may havecertain risk factors for osteoporosis, such as being female, Caucasian,of Asian descent, having a family history of osteoporosis, having afamily history of fractures, and/or having a small body frame.

In accordance with some embodiments of the invention, nitric oxide gasitself may be entrapped or contained within various compositions asdiscussed herein, for example, in liquid crystal multilamellarphosphatidylcholine. In addition, in certain embodiments as discussedbelow, the composition may be stable and can be stored for periods oftime with little or no loss or reaction of the nitric oxide containedtherein.

Since nitric oxide is an unstable and reactive gas, entrapment, storage,and release of nitric oxide requires careful formulation in someembodiments of the invention. For example, nitric oxide readily reactswith water to form nitrous acid (HNO₂), and thus, certain embodiments ofthe invention include compositions or phases that are substantially freeof water. As another example, in one set of embodiments, nitric oxidemay be contained within a first phase comprising a lecithin such asphosphatidylcholine, which may be present within a second phasecomprising an emulsifier, such as is discussed herein. Other components,for example, transdermal penetration enhancers, adjuvants, surfactants,lubricants, etc. can also be present in certain cases.

Thus, the compositions of the invention comprise, in certain aspects, aphase comprising phosphatidylcholine and/or other lecithins in whichnitric oxide is contained within or “trapped.” The phosphatidylcholineor lecithin may be contained within a second phase, for example,comprising an emulsifier, which may cause the phosphatidylcholine orlecithin to form vesicles, e.g., micelles or liposomes. Thephosphatidylcholine or lecithin composition can be unilamellar ormultilamellar in some embodiments. In some instances, the presence ofthe second phase causes the phosphatidylcholine or lecithin to form aliquid crystal arrangement.

The nitric oxide is typically gaseous, and may be present within thecomposition as small bubbles and/or bound to lecithins orphosphatidylcholines within the composition. For example, the nitricoxide may be bound to double bonds present in the lecithins orphosphatidylcholines. Phosphatidylcholine is believed to stabilizeand/or contain the nitric oxide. In some cases, stability of thecomposition can be achieved at room temperature (about 25° C.), and/orat other temperatures such as those described herein. Without wishing tobe bound by any theory, it is believed that the phosphatidylcholineadopts a liquid crystal structure under such conditions, which canthereby contain the nitric oxide, e.g., as small gaseous bubbles, and/orthrough binding with lecithins or phosphatidylcholines.

Nitric oxide is typically reactive with water (e.g., forming nitrousacid), which contributes to its relatively short lifetime within thebody or within other aqueous environments. Accordingly, in certainembodiments of the invention, the composition, or at least a phase ofthe composition comprising the nitric oxide (and/or the second phase,and/or one or more materials used to prepare a nitric oxide composition,and/or a nitric oxide composition prepared as described herein), issubstantially free of water, e.g., comprising no more than about 10 wt%, no more than about 3 wt %, no more than about 1 wt %, no more thanabout 0.3 wt %, or no more than about 0.1 wt % water (i.e., relative tothe weight of the overall composition). The composition may also have nomore than about 1,000 ppm, no more than about 750 ppm, no more thanabout 500 ppm, no more than about 400 ppm, no more than about 300 ppm,no more than about 250 ppm, no more than about 200 ppm, no more thanabout 150 ppm, no more than about 100 ppm, no more than about 50 ppm, nomore than about 25 ppm, or no more than about 10 ppm of water. Incertain embodiments, no detectable water may be present in thecomposition, or at least within a phase of the composition comprisingthe nitric oxide. Any suitable technique can be used for determining theamount of water present in the composition, for example, Karl-Fishertitration. In some cases, the composition may also be free of anyliquids that typically contain water, e.g., physiological buffers, bodyfluids, saline, or the like.

Any suitable amount of nitric oxide may be present within a compositionprepared as described herein. For example, at least about 0.3 wt %, atleast about 0.5 wt %, at least about 0.7 wt %, at least about 1 wt %, atleast about 1.5 wt %, at least about 2 wt %, at least about 2.5 wt %, atleast about 3 wt %, at least about 5 wt % at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, at least about 90 wt %, at least about 100 wt %, atleast about 110 wt %, or at least about 120 wt % of the composition canbe nitric oxide, where the basis of the weight percentage is the weightof the composition before nitric oxide is added. For example, the nitricoxide may be present at between 70 wt % and about 120 wt % of thecomposition. In some embodiments, the nitric oxide may be present at aconcentration of at least about 400 mg/kg, at least about 450 mg/kg, atleast about 500 mg/kg, at least about 550 mg/kg, at least about 570mg/kg, at least about 600 mg/kg, at least about 650 mg/kg, at leastabout 700 mg/kg, at least about 750 mg/kg, at least about 800 mg/kg, atleast about 850 mg/kg, at least about 950 mg/kg, or at least about 1000mg/kg of the composition. In certain cases, the nitric oxide may bepresent at a concentration of no more than about 2000 mg/kg, no morethan about 1500 mg/kg, no more than about 1000 mg/kg, no more than about960 mg/kg, no more than about 900 mg/kg, no more than about 800 mg/kg,no more than about 700 mg/kg, or no more than about 600 mg/kg. Forexample, the nitric oxide may be present at a concentration of betweenabout 570 mg/kg and about 960 mg/kg.

In some embodiments, the nitric oxide is present at a concentration(e.g., on a per-mass basis) of at least about 100 ppm, at least about200 ppm, at least about 300 ppm, at least about 400 ppm, at least about500 ppm, at least about 600 ppm, at least about 700 ppm, at least about800 ppm, at least about 900 ppm, at least about 1000 ppm, at least about1100 ppm, at least about 1200 ppm, at least about 1300 ppm, at leastabout 1400 ppm, at least about 1500 ppm, at least about 1600 ppm, atleast about 1700 ppm, at least about 1800 ppm, at least about 1900 ppm,at least about 2000 ppm, at least about 2500 ppm, at least about 3000ppm, at least about 3500 ppm, at least about 4000 ppm, at least about4500 ppm, at least about 5000 ppm, at least about 6000 ppm, at leastabout 7000 ppm, at least about 8000 ppm, at least about 9000 ppm, or atleast about 10000 ppm of the composition. In other embodiments, thenitric oxide is present at a concentration of no more than about 11000ppm, no more than about 10000 ppm, no more than about 9000 ppm, no morethan about 8000 ppm, no more than about 7000 ppm, no more than about6000 ppm, no more than about 5000 ppm, no more than about 4500 ppm, nomore than about 4000 ppm, no more than about 3500 ppm, no more thanabout 3000 ppm, no more than about 2500 ppm, no more than about 2000ppm, no more than about 1900 ppm, no more than about 1800 ppm, no morethan about 1700 ppm, no more than about 1600 ppm, no more than about1500 ppm, no more than about 1400 ppm, no more than about 1300 ppm, nomore than about 1200 ppm, no more than about 1100 ppm, no more thanabout 1000 ppm, no more than about 900 ppm, no more than about 800 ppm,no more than about 700 ppm, no more than about 600 ppm, no more thanabout 500 ppm, no more than about 400 ppm, or no more than about 300 ppmof the composition. For example, in some embodiments, nitric oxide ispresent at a concentration of between about 400 and about 900 ppm. NOcontent can be measured by any suitable technique. For example, in someembodiments, NO content is measured using a nitric oxide biosensor(e.g., nitric oxide macrosensor with nitric oxide specific electrodefrom WPI Instruments). In some embodiments, NO content is measured by achange in weight in the composition after adding NO.

In some embodiments, nitric oxide is present within a first phasecomprising a lecithin, such as phosphatidylcholine. Phosphatidylcholine(herein abbreviated “PC”) is a basic component of cell membrane bilayersand the main phospholipid circulating in the plasma of blood.Phosphatidylcholine typically has a phospholipid structure with acholine head group and a glycerophosphoric acid tail group. The tailgroup can be saturated or unsaturated. More than one tail group may bepresent in the phosphatidylcholine in some cases, and the tail groupsmay be the same or different. Specific non-limiting examples ofphosphatidylcholines that could be used include one or a mixture ofstearic, palmitic, margaric, and/or oleic acid diglycerides linked to acholine ester head group.

Phosphatidylcholines are a member of a class of compounds calledlecithins. Typically, a lecithin is a composed of phosphoric acid,choline, fatty acids, glycerol, glycolipids, triglycerides, and/orphospholipids. In some cases, other lecithins may be used, in additionto or instead of a phosphatidylcholine. Non-limiting examples of otherlecithins include phosphatidylethanolamine, phosphatidylinositol, orphosphatidic acid. Many commercial lecithin products are available, suchas, for example, Lecithol®, Vitellin®, Kelecin®, and Granulestin®.Lecithin is widely used in the food industry. In some embodiments,certain compositions of the invention can contain synthetic or naturallecithin, or mixtures thereof. Natural preparations are used in somecases because they exhibit desirable physical characteristics, and/ormay be economical or nontoxic. However, in other embodiments,non-natural preparations are used, or the composition can include bothnatural and non-natural preparations.

Any suitable amount of phosphatidylcholine or lecithin may be presentwithin the composition. For example, at least about 0.25 wt %, at leastabout 0.5 wt %, at least about 1 wt %, at least about 2 wt %, at leastabout 3 wt %, at least about 5 wt %, at least about 8 wt %, at leastabout 10 wt %, at least about 20 wt %, at least about 30 wt %, at leastabout 40 wt %, at least about 50 wt %, at least about 60 wt %, at leastabout 70 wt %, at least about 80 wt %, or at least about 90 wt % of theentire composition can be a phosphatidylcholine or a lecithin. In somecases, the phosphatidylcholine or lecithin may be present at aconcentration of no more than about 95 wt %, no more than about 90 wt %,no more than about 80 wt %, no more than about 70 wt %, no more thanabout 65 wt %, no more than about 60 wt %, no more than about 50 wt %,no more than about 40 wt %, no more than about 30 wt %, no more thanabout 20 wt %, or no more than about 10%. For instance, thephosphatidylcholine or lecithin may be present at between about 8 wt %and about 65 wt %, or between about 0 wt % and about 10 wt %, etc. Oneor more than one type of phosphatidylcholine or lecithin may be present.

Some delivery compositions of the present invention may containpolyenylphosphatidylcholine (herein abbreviated “PPC”). In some cases,PPC can be used to enhance epidermal penetration. The term“polyenylphosphatidylcholine,” as used herein, means anyphosphatidylcholine bearing two fatty acid moieties, wherein at leastone of the two fatty acids is an unsaturated fatty acid with at leasttwo double bonds in its structure, such as linoleic acid.

Certain types of soybean lecithin and soybean fractions, for example,can contain higher levels of polyenylphosphatidylcholine, withdilinoleoylphosphatidylcholine (18:2-18:2 phosphatidylcholine) as themost abundant phosphatidylcholine species therein, than conventionalfood grade lecithin. Such lecithins may be useful in formulating certaindelivery compositions. In some embodiments, conventional soybeanlecithin may be enriched with polyenylphosphatidylcholine, for instance,by adding soybean extracts containing high levels ofpolyenylphosphatidylcholine. As used herein, this type ofphosphatidylcholine is called “polyenylphosphatidylcholine-enriched”phosphatidylcholine (hereinafter referred to as PPC-enrichedphosphatidylcholine), even where the term encompasses lecithin obtainedfrom natural sources exhibiting polyenylphosphatidylcholine levelshigher than ordinary soybean varieties. These products are commerciallyavailable, for example, from American Lecithin Company, Rhone-Poulencand other lecithin vendors. American Lecithin Company markets itsproducts with a “U” designation, indicating high levels of unsaturation;Rhone-Poulenc's product is a soybean extract containing about 42%dilinoleoylphosphatidylcholine and about 24%palmitoyllinoleylphosphatidylcholine (16:0 to 18:2 of PC) as the majorphosphatidylcholine components. Another example of a suitablepolyenylphosphatidylcholine is NAT 8729 (also commercially availablefrom vendors such as Rhone-Poulenc and American Lecithin Company).

Any suitable amount of polyenylphosphatidylcholine may be present withinthe composition. For example, at least about 0.25 wt %, at least about0.5 wt %, at least about 1 wt %, at least about 2 wt %, at least about 3wt %, at least about 5 wt %, at least about 8 wt %, at least about 10 wt%, at least about 20 wt %, at least about 30 wt %, at least about 40 wt%, at least about 50 wt %, at least about 60 wt %, at least about 70 wt%, at least about 80 wt %, or at least about 90 wt % of the compositioncan be polyenylphosphatidylcholine. In some cases, thepolyenylphosphatidylcholine may be present at a concentration of no morethan about 95 wt %, no more than about 90 wt %, no more than about 80 wt%, no more than about 70 wt %, no more than about 65 wt %, no more thanabout 60 wt %, no more than about 50 wt %, no more than about 40 wt %,no more than about 30 wt %, no more than about 20 wt %, or no more thanabout 10%. For instance, the polyenylphosphatidylcholine may be presentat between about 8 wt % and about 65 wt %. In some embodiments, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, at leastabout 50 wt %, at least about 60 wt %, at least about 70 wt %, at leastabout 80 wt %, at least about 90 wt %, or about 100 wt % of all of thephosphatidylcholine or lecithin in the composition ispolyenylphosphatidylcholine.

While not wishing to be bound to any theory, it is believed that thePPC-enriched phosphatidylcholine forms a bilayer enveloping nitric oxide(and in some embodiments, other adjunct ingredients, if present) tocreate the drug delivery composition. The PPC-enrichedphosphatidylcholine is believed to contribute to the stability of thenitric oxide, for example, by shielding the nitric oxide from water,and/or by enhancing its penetration into the skin or other area, e.g., amucosal surface.

The first phase also comprises, in some embodiments of the invention, afatty acid ester. Non-limiting examples include ascorbate palmitate orisopropyl palmitate. In some cases, the fatty acid ester is used as apreservative or an antioxidant. The composition can include any suitableamount of fatty acid ester, for example, at least about 1 wt %, at leastabout 3 wt %, at least about 5 wt %, at least about 10 wt %, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, at leastabout 50 wt %, etc. In some cases, no more than about 60 wt %, no morethan about 50 wt %, no more than about 40 wt %, no more than about 30 wt%, no more than about 20 wt %, no more than about 18 wt %, no more thanabout 15 wt %, no more than about 12 wt %, or no more than about 10 wt %of the composition is fatty acid ester. For example, the composition maybe between about 0 wt % and about 10 wt % fatty acid ester. Thecomposition may include one or more than one fatty acid ester.

In certain embodiments, a drug delivery composition such as thosedescribed herein can be formulated to include a second phase. Typically,the second phase is substantially immiscible with the first phasecomprising phosphatidylcholine or lecithin. Two phases that aresubstantially immiscible are able to form discrete phases when exposedto each other at ambient conditions (e.g., 25° C. and 1 atm) forextended periods of time (e.g., at least about a day). The phases can beseparate identifiable phases (e.g., one may float above the other), orin some cases, the phases are intermingled, e.g., as in an emulsion. Thestability of the discrete phases may be kinetic and/or thermodynamic innature, in various embodiments.

In one set of embodiments, the second phase may comprise an emulsifierwhich causes the first phase comprising phosphatidylcholine or lecithinto form a liquid crystal, and/or vesicles such as micelles or liposomes.Typically, in a liquid crystal phase, vesicular structures such asmicelles, liposomes, hexagonal phases, or lipid bilayers can be formed.In some cases, multilamellar structures may be present within the liquidcrystal phase, although in other cases, only unilamellar structures maybe present. For example, in certain cases, the PPC-enrichedphosphatidylcholine can be loosely arranged in a multilamellar fashion,with nitric oxide and optional adjunct ingredients being bonded orotherwise entrapped or contained within the lipid bilayers formedtherein. In some cases, the first phase (e.g., comprising PPC-enrichedphosphatidylcholine) and the second phase can form a structure such asis disclosed in U.S. Pat. No. 7,182,956 to Perricone, et al. This isbelieved (without wishing to be bound by any theory) to form a looselyarranged, yet stable, PPC-enriched phosphatidylcholine-drug complex thatmay allow penetration and delivery of nitric oxide and optional adjunctingredients to the skin, e.g., to the dermal vasculature.

In one set of embodiments, the second phase comprises an emulsifier. Theemulsifier, in one embodiment, is a substance that is able to stabilizean emulsion by increasing its kinetic stability. The emulsifier may alsobe chosen in some cases to be relatively inert or non-toxic relative tothe skin.

In some embodiments, the second phase may comprise a polyglycol. Thepolyglycol may include a polyhydric alcohol of a monomeric glycol suchas polyethylene glycol (PEG) and/or polypropylene glycol (PPG). Forexample, the PEG or PPG may be PEG or PPG 200, 300, 400, 600, 1,000,1,450, 3,350, 4,000, 6,000, 8,000, and 20,000, where the numberindicates the approximate average molecular weight of the PEG or PPG. Asis understood by those of ordinary skill in the art, a polyglycolcomposition often will comprise a range of molecular weights, althoughthe approximate average molecular weight is used to identify the typepolyglycol. More than one PEG and/or PPG can also be present in certaininstances.

The second phase may comprise a surfactant in some embodiments.Non-limiting examples of surfactants include a siloxylated polyethercomprising dimethyl, methyl(propylpolyethylene oxide propylene oxide,acetate) siloxane commercially available from vendors such as DowCorning (Dow Corning 190 surfactant). Other examples of materials thatcan be used as (or within) the second phase include, but are not limitedto, 1,2-propanediol, or silicone fluids containing low viscositypolydimethylsiloxane polymers, methylparaben (p-hydroxy benzoic acidmethyl ester) commercially available from vendors such as Dow Corning(Dow Corning 200 silicone fluid). Still other examples include varioussiloxane or silicone compounds, e.g., hexamethyldisiloxane,amodimethicone, phenyltrimethicone, etc.

Additionally, purified water may be added to the second phase in someembodiments, although in other cases, little or no water is present inthe second phase. For example, the first phase, the second phase, cancontain less than 10%, less than 5%, less than 2%, less than 1%, or lessthat 0.05% (e.g., wt %) of water relative to the weight of therespective phase or of the entire composition. In some cases, the secondphase may also comprise adjunct ingredients such as those describedherein.

The second phase may include any one, or more than one, of the materialsdescribed above. In addition, any suitable amount of second phase can beused in accordance with various embodiments of the invention. Forexample, the second phase may be present at at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, or at least about 90 wt % of the composition. Insome cases, the ratio of the first phase (e.g., comprisingphosphatidylcholine or lecithin) to the second phase can be at leastabout 1:3, at least about 1:2, at least about 1:1, at least about 2:1,at least about 3:1, or at least about 4:1, etc.

In another set of embodiments, the composition may also include one ormore transdermal penetration enhancers. Examples of transdermalpenetration enhancers include, but are not limited to,1,3-dimethyl-2-imidazolidinone or 1,2-propanediol. Other examplesinclude cationic, anionic, or nonionic surfactants (e.g., sodium dodecylsulfate, polyoxamers, etc.); fatty acids and alcohols (e.g., ethanol,oleic acid, lauric acid, liposomes, etc.); anticholinergic agents (e.g.,benzilonium bromide, oxyphenonium bromide); alkanones (e.g., n-heptane);amides (e.g., urea, N,N-dimethyl-m-toluamide); organic acids (e.g.,citric acid); sulfoxides (e.g., dimethylsulfoxide); terpenes (e.g.,cyclohexene); ureas; sugars; carbohydrates or other agents. Thetransdermal penetration enhancers can be present in any suitable amountwithin the composition. For example, at least about 10 wt %, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, or atleast about 50 wt % of the composition may comprise one or moretransdermal penetration enhancers. In some cases, no more than about 60wt %, no more than about 50 wt %, no more than about 40 wt %, no morethan about 30 wt %, no more than about 20 wt %, no more than about 10 wt%, no more than about 9 wt %, or no more than about 5 wt % of thecomposition comprises transdermal penetration enhancers. For example,the composition may have between about 0 wt % and about 5 wt % of one ormore transdermal penetration enhancers.

As a specific non-limiting example of one set of embodiments, apolyenylphosphatidylcholine comprises a certain material with the tradename NAT 8729, and optionally at least one polyglycol (polyhydricalcohol of a monomeric glycol such as polyethylene glycol 200, 300, 400,600, 1,000, 1,450, 3,350, 4,000, 6,000, 8,000 and 20,000). Thecomposition can also comprise a PPC-enriched phosphatidylcholinematerial that is present within the first or second phase, e.g.,comprising nitric oxide. The second phase may also comprise a surfactantsuch as a siloxylated polyether comprising dimethyl,methyl(propylpolyethylene oxide propylene oxide, acetate) siloxanecommercially available from vendors such as Dow Corning (Dow Corning 190surfactant) and lubricant such as silicone fluids containing lowviscosity polydimethylsiloxane polymers, methylparaben (p-hydroxybenzoic acid methyl ester) commercially available from vendors such asDow Corning (Dow Corning 200 silicone fluid).

In some embodiments, various compositions of the invention areformulated to be substantially clear or substantially transparent.Transparency may be useful, for instance, for product acceptance in themarketplace, e.g., when applied to the skin of a subject. However, inother embodiments, the composition is not necessarily transparent.Certain substances can be useful in providing a substantiallytransparent composition, for example, fatty acid esters such asascorbate palmitate or isopropyl palmitate. In one set of embodiments,the composition may be substantially transparent such that incidentvisible light (e.g., have wavelengths of between about 400 nm and about700 nm) can be transmitted through 1 cm of the composition with a lossin intensity of no more than about 50%, about 60%, about 70%, about 80%,or about 90% relative to the incident light. In some embodiments, theremay be no substantial difference in the wavelengths that are absorbed bythe composition (i.e., white light passing through the compositionappears white), although in other cases, there can be more absorption atvarious wavelengths (for example, such that white light passing throughthe composition may appear colored).

Other components may also be present within the composition, inaccordance with certain embodiments of the invention. For example, thecomposition may include volatile organic fluids, fatty acids, volatilearomatic cyclic compounds, high molecular weight hydrocarbons, or thelike.

In accordance with certain aspects of the invention, the composition maybe prepared by mixing a first phase and a second phase together, thenpassing nitric oxide through the mixture. As discussed above, the secondphase can comprise an emulsifier, or any other components discussedherein. The first phase may comprise a lecithin such asphosphatidylcholine and/or polyenylphosphatidylcholine, e.g.,PPC-enriched phosphatidylcholine, for instance, as described herein. Insome embodiments, other components are also mixed into the composition,before or after (or while) adding nitric oxide, for example, transdermalpenetration enhancers, adjuvants, polyglycols (e.g., PEG and/or PPG),surfactants, lubricants, etc. as discussed herein. In some embodiments,however, nitric oxide may be passed through the first phase prior tomixing of the first phase with the second phase.

In one set of embodiments, after forming the mixture, nitric oxide canbe passed into or through the mixture, for example, by blowing bubblesof nitric oxide through the mixture. Nitric oxide may be delivered intothe mixture under pressures such as between about 3,000 Pa and about15,000 Pa, between about 5,000 Pa and about 10,000 Pa, or between about6,000 Pa and about 8,000 Pa, and/or temperatures such as between about0° C. and about 50° C., between about 20° C. and about 35° C., or about25° C. and about 30° C. However, higher or lower pressures also may beused in some embodiments as aspects of the invention are not limited inthis respect.

In certain embodiments, the nitric oxide is bubbled through the mixtureuntil the mixture begins to at least partially solidify. As an example,the viscosity of the mixture may increase to at least about 1,000 cP, atleast about 2,000 cP, at least about 3,000 cP, at least about 5,000 cP,at least about 7,000 cP, at least about 10,000 cP, at least about 12,000cP, at least about 15,000 cP, at least about 20,000 cP, at least about30,000 cP, at least about 40,000 cP, at least about 50,000 cP, at leastabout 60,000 cP, at least about 70,000 cP, or at least about 80,000 cP.The nitric oxide can be passed through the mixture as pure nitric oxide,and/or with other gases (e.g., a noble gas, for example, argon). In somecases, a nitric oxide donor may be passed into the mixture, and therein,at least some of the nitric oxide donor can be converted into nitricoxide. In other embodiments, however, the final composition may havelower viscosities, for example, such that the composition is liquid, orcould be sprayed onto the skin.

In one set of embodiments, the nitric oxide can be bubbled through themixture to cause the viscosity of the mixture to increase. For example,the viscosity can increase until the mixture begins to form a gel, acream, a lotion, an ointment, a solid “stick,” or the like. A cream maybe, for example, a semi-solid emulsion, e.g., comprising a first phaseand a second phase. The first phase may be discontinuous (e.g.,comprising small droplets or vesicles, such as is discussed herein) andthe second phase may be continuous, or vice versa. In some cases,however, both the first phase and the second phase are co-continuouswithin the mixture.

In some embodiments of the invention, a composition may be prepared asdiscussed above, then diluted, e.g., with a diluent, to produce a finalcomposition. For example, a “stock” composition may be initiallyprepared, e.g., having a relatively high nitric oxide concentration,then the stock composition diluted to produce a final composition, e.g.,before use, before storage, before packaging, etc. In some embodiments,the diluent used may be a component as discussed herein (for example,forming at least a portion of the second phase), and the same ordifferent materials than may be present in the initial composition maybe used. The dilution ratio (amount of diluent added, relative to theinitial composition) may be at least about 2, at least about 3, at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 50, or at least about100, or any other suitable factor.

A composition may be prepared and/or stored at any suitable temperatureand under any suitable conditions. In some embodiments, for instance, acomposition can be prepared and/or stored under limited or no oxygenconditions, as oxygen can adversely react with nitric oxide. Thecomposition can also be prepared and/or stored under limited or nonitrogen and/or carbon dioxide, as both can also react adversely withnitric oxide. For instance, the composition may be prepared and/orstored in a sealed environment (e.g., stored in a sealed container). Thesealed environment (e.g., container) can be at least substantiallydevoid of gas, and/or contains a gaseous mixture that excludes, or atleast is depleted in, oxygen. In some embodiments, an environmentdepleted in oxygen may have less than about 20%, less than about 15%,less than about 10%, less than about 5%, about 1% or less, about 0.1% orless, about 0.01% or less, about 0.001% or less, oxygen (e.g., as a wt %or as molar % per volume). For example, the gaseous mixture may includea noble gas, such as argon, helium, neon, etc. In one set ofembodiments, the container may comprise a multi-layered metallic and/orpolymeric barrier, e.g., formed from Glaminate® (American Can Company).For instance, the container may have the shape of a tube. Thus, incertain embodiments, the container is substantially resistant to oxygenpermeation, nitrogen permeation, and/or carbon dioxide permeation. Incertain embodiments, the container is substantially watertight, forexample, such that substantially no water is absorbed by the container,or such that no water is able to pass through the container even if thecontainer is filled with water.

As previously discussed, nitric oxide can react with water, and thus,compositions described herein may be prepared and/or stored underconditions where substantially no water is present. For example, nitricoxide and/or a nitric oxide containing preparation described herein maybe prepared and/or stored under relatively low relative humidities(e.g., less than about 50% RH, less than about 40% RH, less than about30% RH, less than about 20% RH, or less than about 10% RH), and/or inthe presence of a suitable desiccant, such as phosphorous pentoxide orsilica gel.

In certain embodiments, the mixture may be mixed with or otherwiseinclude adjunct ingredients, if applicable, and nitric oxide may beintroduced to the mixture, e.g., bubbles of nitric oxide gas may beblown into the mixture until the mixture hardens to obtain the desiredfinal composition. As a specific non-limiting example, a nitric oxidecomposition may be formed by preparing a non-liposome multilamellarliquid crystal phosphatidylcholine phase, for example, by providing apolyglycol, then introducing phosphatidyl choline into the glycol atroom temperature to form a phosphatidylcholine solution. Thephosphatidylcholine often comes as a solid (e.g., as a “brick” ofmaterial), and the phosphatidylcholine may be broken down into smallerpieces to aid in mixing, e.g., by “shaving” or grinding thephosphatidylcholine solid. The phosphatidylcholine solution is mixeduntil the phosphatidylcholine solution is substantially clear, then onemay warm the phosphatidylcholine solution to 40° C., mill the warmedsolution (i.e., low agitation after the initial mixing), combinesiloxylated polyether and polydimethylsiloxane to form a fluid, add thefluid to the warmed solution and milling until the solution is clear,adding methyl paraben or other suitable lubricant to the solution andmilling until the methyl paraben dissolves in the solution, warm waterto 40° C. and adding the warmed water slowly to the solution, and thenceasing milling of the solution and “sweeping” the solution (e.g., witha sweep mixer) to cool to room temperature. Nitric oxide gas can then bebubbled or otherwise introduced into the solution while cooling thesolution until the solution begins to harden or becomes stiff, e.g.,having the consistency of a gel or a cream, such as previouslydescribed. In some cases, the resulting composition is sealed in acontainer, for example, as discussed herein. Any suitable container maybe used, e.g., a tube or a bottle. In addition, the composition (e.g.,within the container) may be stored at room temperature, or any othersuitable temperature. For example, a composition of the invention may bestored at or below 80° C., e.g., at or below room temperature (about 25°C.) or in a refrigerator (e.g., at 4° C.) for extended period ofstorage, for instance, to prevent nitric oxide leakage or denaturing. Insome cases, storage may extend for at least about a week, at least about4 weeks, at least about 6 months, at least about a year, etc.

It is surprising that, according to some embodiments, nitric oxide notonly can be entrapped in phosphatidylcholine or lecithin compositionssuch as those described herein, but also that such entrappedcompositions may have a long shelf life, especially when refrigerated.No loss or reaction of nitric oxide is expected during extendedrefrigerated storage, at least under certain conditions. For instance,in certain embodiments, the composition may be stored at temperatures ofless than about 80° C., less than about 70° C., less than about 60° C.,less than about 50° C., less than about 40° C., less than about 30° C.,less than about 25° C., less than about 20° C., less than about 15° C.,less than about 10° C., less than about 5° C., less than about 0° C.,etc., for extended periods of time, e.g., at least about a day, at leastabout a week, at least about 4 weeks, at least about 6 months, etc.

Without wishing to be bound by theory, it is believed that nitric oxideforms reversible physical bonds, similar to hydrogen bonds or van derWaals forces, with phosphatidylcholine or other lecithin molecules,e.g., containing one or more double bonds, which may allow nitric oxideto become entrapped and thereby remain intact for an extended period oftime, e.g., during storage. These physical bonds, however, are believedto be not very stable, and may in some cases be easily broken up, forexample, upon various physical agitations such as rubbing thecomposition against the skin, thereby releasing the entrapped nitricoxide. While others have stabilized other substances or drugs withinphosphatidylcholine or lecithin compositions or vesicles, for example,protein drugs such as insulin, it is surprising that a small, highlyreactive molecule such as NO could similarly be stabilized, especiallywhen it would have been expected that a molecule as small as NO wouldreadily diffuse away from such compositions and/or would have reactedwith water that is typically present within such compositions.

In some embodiments, it is believed that other species reactive withwater could also be similarly stabilized, e.g., within a composition asherein described. Any species that ordinarily reacts with water could bestabilized within such compositions. Examples of such species include,but are not limited to, lithium, or drugs or polymers with labile bondssusceptible to hydrolysis, for instance, certain peptides,polysaccharides, polylactic acid, polyglycolic acid, etc.

In certain aspects of the invention, a composition such as thosedescribed herein can be administered to a subject, such as a humansubject, by rubbing it on the skin of the subject, e.g., in areaslocated at or at least within the vicinity of a desired target area.Without wishing to be bound by any theory, it is believed thatphosphatidylcholine provides or facilitates delivery of nitric oxide tothe skin, allowing nitric oxide to be delivered to a target area. Insome embodiments, the composition can be applied, by rubbing thecomposition topically against the skin, which allows the composition (orat least, nitric oxide) to be absorbed by the skin. The composition canbe applied once, or more than once. For example, the composition may beadministered at predetermined intervals. In some embodiments, forinstance, the composition may be applied once per day, twice per day, 3times per day, 4 times per day, once every other day, once every threedays, once every four days, etc. The amount of nitric oxide necessary tobring about the therapeutic treatment is not fixed per se, and maydepend upon factors such as the desired outcome, the type and severitythe disease or condition, the form of nitric oxide, the concentration ofnitric oxide present within the composition, etc.

Thus, another aspect of the invention provides methods of administeringany composition such as discussed herein to a subject. The compositionsof the invention may be applied to a subject for local or systemicdelivery, depending on the application. The compositions of theinvention may also be applied to any suitable area of the skin. Forexample, the compositions may be applied to the upper chest or arms, orto any hairy or non-hairy portion of the skin, e.g., to promote systemicdelivery of nitric oxide, or the composition may be applied locally,e.g., at an area of the skin where treatment is desired.

When administered, the compositions of the invention are applied in atherapeutically effective, pharmaceutically acceptable amount as apharmaceutically acceptable formulation. Any of the compositions of thepresent invention may be administered to the subject in atherapeutically effective dose. When administered to a subject,effective amounts will depend on the particular condition being treatedand the desired outcome. A therapeutically effective dose may bedetermined by those of ordinary skill in the art, for instance,employing factors such as those described herein and using no more thanroutine experimentation.

In some embodiments, an effective amount is an amount sufficient to havea measurable positive effect on blood flow and/or vasodilation, and/or ameasurable negative effect on blood pressure. In some embodiments, theeffect on blood flow and/or vasodilation is observed local to the siteof topical application. In some embodiments, an effective amount is anamount sufficient to have a measurable effect on osteoporosis asevidenced by an appropriate clinical parameter, e.g., reduction innumber of new fractures (e.g., vertebral fractures), increase in bonedensity. maintenance of bone density. In some embodiments, an effectiveamount is an amount sufficient to obtain a systemic level of nitricoxide that is sufficient to have a desired effect, e.g., have ameasurable positive effect on blood flow and/or vasodilation, have ameasurable negative effect on blood pressure, and/or have a measurableeffect on osteoporosis as evidenced by an appropriate clinicalparameter.

The compositions described herein can be used in combination therapywith one or more additional therapeutic agents. For combinationtreatment with more than one active agent, where the active agents arein separate dosage formulations, the active agents may be administeredseparately or in conjunction. In addition, the administration of oneelement may be prior to, concurrent to, or subsequent to theadministration of the other agent. In certain embodiments, theadditional therapeutic agent is present in a provided composition inaddition to nitric oxide. In other embodiments, the additionaltherapeutic agent is administered separately from the nitric oxidecontaining composition.

When co-administered with other agents, e.g., when co-administered withanother anti-osteoporosis medication, an “effective amount” of thesecond agent will depend on the type of drug used. Suitable dosages areknown for approved agents and can be adjusted by the skilled artisanaccording to the condition of the subject, the type of condition(s)being treated and the amount of a compound described herein being used.In cases where no amount is expressly noted, an effective amount shouldbe assumed. For example, compounds described herein can be administeredto a subject in a dosage range from between about 0.01 to about 10,000mg/kg body weight/day, about 0.01 to about 5000 mg/kg body weight/day,about 0.01 to about 3000 mg/kg body weight/day, about 0.01 to about 1000mg/kg body weight/day, about 0.01 to about 500 mg/kg body weight/day,about 0.01 to about 300 mg/kg body weight/day, about 0.01 to about 100mg/kg body weight/day.

In one set of embodiments, the dosage may be between about 0.01 mg andabout 500 g, between about 0.01 mg and about 300 g, between about 0.01mg and about 100 g, between about 0.01 mg and about 30 g, between about0.01 mg and about 10 g, between about 0.01 mg and about 3 g, betweenabout 0.01 mg and about 1 g, between about 0.01 mg and about 300 mg,between about 0.01 mg and about 100 mg, between about 0.01 mg and about30 mg, between about 0.01 mg and about 10 mg, between about 0.01 mg andabout 3 mg, between about 0.01 mg and about 1 mg, between about 0.01 mgand about 0.3 mg, or between about 0.01 mg and about 0.1 mg.

In certain embodiments, a nitric oxide containing composition asdescribed herein, and the additional therapeutic agent are eachadministered in an effective amount (i.e., each in an amount which wouldbe therapeutically effective if administered alone). In otherembodiments, a nitric oxide containing composition as described herein,and the additional therapeutic agent are each administered in an amountwhich alone does not provide a therapeutic effect (a sub-therapeuticdose). In yet other embodiments, a nitric oxide containing compositionas described herein can be administered in an effective amount, whilethe additional therapeutic agent is administered in a sub-therapeuticdose. In still other embodiments, a nitric oxide containing compositionas described herein can be administered in a sub-therapeutic dose, whilethe additional therapeutic agent is administered in an effective amount.

As used herein, the terms “in combination” or “co-administration” can beused interchangeably to refer to the use of more than one therapy (e.g.,one or more prophylactic and/or therapeutic agents). The use of theterms does not restrict the order in which therapies (e.g., prophylacticand/or therapeutic agents) are administered to a subject.

Co-administration encompasses administration of the first and secondamounts of the compounds in an essentially simultaneous manner, such asin a single pharmaceutical composition, for example, capsule or tablethaving a fixed ratio of first and second amounts, or in multiple,separate capsules or tablets for each. In addition, suchco-administration also encompasses use of each compound in a sequentialmanner in either order. When co-administration involves the separateadministration of the first amount of a composition as described herein,and a second amount of an additional therapeutic agent, the compoundsare administered sufficiently close in time to have the desiredtherapeutic effect. For example, the period of time between eachadministration which can result in the desired therapeutic effect, canrange from minutes to hours and can be determined taking into accountthe properties of each compound. For example, a composition as describedherein, and the second therapeutic agent can be administered in anyorder within about 24 hours of each other, within about 16 hours of eachother, within about 8 hours of each other, within about 4 hours of eachother, within about 1 hour of each other or within about 30 minutes ofeach other.

More specifically, a first therapy (e.g., a prophylactic or therapeuticagent such as a composition described herein) can be administered priorto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapy to a subject.

Examples of therapeutic agents that may be combined with a compositionof this disclosure, either administered separately or in the samepharmaceutical composition, include, but are not limited to,bisphosphonates (e.g., alendronate, risedronate, ibandronate, zoledronicacid), hormone-related therapy (e.g., estrogen, raloxifene,testosterone), teriparatide, denosumab, calcitonin, parathyroid hormonepeptides (e.g., Forteo, Ostabolin-C), osteoblast activating peptide,osteocalcin, and osteogenic growth peptide.

In certain embodiments of the invention, the administration of variouscompositions of the invention may be designed so as to result insequential exposures to the composition over a certain time period, forexample, hours, days, weeks, months, or years. This may be accomplished,for example, by repeated administrations of a composition of theinvention by one or more of the methods described herein, or by asustained or controlled release delivery system in which the compositionis delivered over a prolonged period without repeated administrations.Administration of the composition using such a delivery system may be,for example, by a transdermal patch. Maintaining a substantiallyconstant concentration of the composition may be preferred in somecases.

In one set of embodiments, a composition such as is discussed herein maybe applied to the skin of a subject, e.g., at any suitable location. Insome embodiments, the suitable location is at a site where treatment ofosteoporosis is desired. In some embodiments, the suitable location isnear the site where treatment of osteoporosis is desired (e.g., within1-10, 10-30, 20-50 cm). It will be understood by one skilled in the artthat the bone is not at the level of the skin, and the depth ofpenetration of the composition can be adjusted accordingly. In someembodiments, the suitable location is an area that covers the site wheretreatment of osteoporosis is desired and is, for example, about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 cm in diameter. In someembodiments, when the composition is administered for systemic delivery,the suitable location can be anywhere on the body. The composition maybe contacted using any suitable method. For example, the composition maybe rubbed on, poured on, applied with an applicator (e.g., a gauze pad,a swab, a bandage, etc.), or the like. In some cases, the compositioncan be a liquid, a gel, a cream, a lotion, an ointment, a solid “stick,”or the like, that can be applied to the skin by hand, for example, byrubbing or spraying.

In some embodiments, provided compositions are not used for treatingerectile dysfunction or sexual dysfunction. In certain embodiments, asubject of the methods described herein does not suffer from erectiledysfunction or sexual dysfunction. In some embodiments, a providedcomposition is not applied to the skin of a subject on or near a male orfemale genital region to treat sexual dysfunction and/or to enhancesexual performance or experience. In some embodiments, a providedcomposition is not applied to the perineal region (e.g., penis), or tothe vulva of a subject.

In some embodiments, provided compositions are not used to promote woundhealing. In certain embodiments, a subject of the methods describedherein does not suffer from a wound. In certain embodiments, a providedcomposition is not applied to a wound. Examples of wounds include cuts,scrapes, other traumatic wounds, burns or other accidental wounds, e.g.,an anal fissure, a surgical site, a trauma site, a burn, an abrasion, asunburn, a cut or laceration on the skin, or any other damaged region ofthe skin. In some embodiments, the composition is not applied to amucosal surface of the subject, for example, to the nose. In certainembodiments, provided compositions are not used to treat wounds thatresult from surgical intervention (e.g., any medical intervention oroperation that requires wound healing as part of the recovery process).In some embodiments, a composition of the invention is not used toprepare a tissue site for wound healing prior to the wound (e.g., priorto surgery) and/or after the wound (e.g., and/or after surgery). In someembodiments, a composition of the invention is not applied to thesurface of a wound or to skin prior to a wound.

In some embodiments, a composition of the invention is not applied to asurgical device, tool, or other substrate, such as sutures, implants,surgical tools, surgical dressings, bandages, or other substrates thatmay come into contact with wounded tissue during surgery. In someembodiments, compositions of the invention may be provided as a cream orointment as described in more detail herein.

In some embodiments, a composition of the present invention is not usedto promote hair growth. In some embodiments, a provided composition isnot applied in a region on a subject where hair loss is undesirable,such as the skin surface of the scalp. In some embodiments, a subject ofmethods provided herein is not a subject who is bald, balding, or hasthinning hair or may be at risk of losing hair.

Compared to other means of administration, the use of topicaladministration in certain embodiments of the present invention hasvarious advantages, including one or more of the following. In somecases, administration of a composition and delivery of nitric oxide asdiscussed herein is easier and more effective than other drugadministration routes, for example, oral delivery. Unlike oraladministration where a substantial amount of nitric oxide may bedestroyed during the digestive process, nitric oxide delivered topicallyis not exposed to the digestive tract. Topical application may alsoallow, in some instances, relatively steady delivery of nitric oxide tothe desired target area without the cyclic dosages typical of orally orparenterally administered drugs. In some embodiments, topicalapplication may also avoid toxic side effects associated with sustainedincreased levels of nitric oxide typical of oral or parenteraladministration.

Compared to other topical delivery systems that employ nitric oxidedonors (an entity that is able to release nitric oxide, such asL-arginine, nitroglycerin, or amyl nitrite) as a nitric oxide source,various aspects of the present invention utilizing nitric oxide gas haveseveral advantages, including one or more of the following. Nitric oxidecan be released relatively quickly in some embodiments, because therelease does not necessarily involve chemical transformations of nitricoxide donors to release nitric oxide. The concentration of nitric oxidecan accumulate quickly upon topical administration, leading to goodtherapeutic effect in certain embodiments of the invention. In someembodiments, the release rate of nitric oxide can be controlled, forinstance, by physical actions (e.g., by controlling how much of thecomposition is applied to the skin), in comparison to nitric oxidedonors which release nitric oxide upon chemical stimulation. Moreover,certain embodiments of the present invention employ phosphatidylcholine,a component of cell membranes, as a carrier which improves thepenetration and absorption of nitric oxide into cells and tissues. Thus,certain compositions of the present invention will be non-toxic orbiocompatible.

The compositions of the present invention may additionally comprise oneor more adjunct ingredients, for instance, pharmaceutical drugs, skincare agents, and/or excipients. For example, compositions of theinvention may include additional ingredients such as salts, bufferingagents, diluents, excipients, chelating agents, fillers, drying agents,antioxidants, antimicrobials, preservatives, binding agents, bulkingagents, silicas, solubilizers, or stabilizers. Non-limiting examplesinclude species such as calcium carbonate, sodium carbonate, lactose,kaolin, calcium phosphate, or sodium phosphate; granulating anddisintegrating agents such as corn starch or algenic acid; bindingagents such as starch, gelatin or acacia; lubricating agents such asmagnesium stearate, stearic acid, or talc; time-delay materials such asglycerol monostearate or glycerol distearate; suspending agents such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone;dispersing or wetting agents such as lecithin or othernaturally-occurring phosphatides; thickening agents such as cetylalcohol or beeswax; buffering agents such as acetic acid and saltsthereof, citric acid and salts thereof, boric acid and salts thereof, orphosphoric acid and salts thereof; or preservatives such as benzalkoniumchloride, chlorobutanol, parabens, or thimerosal. Suitableconcentrations can be determined by those of ordinary skill in the art,using no more than routine experimentation. Those of ordinary skill inthe art will know of other suitable formulation ingredients, or will beable to ascertain such, using only routine experimentation.

Preparations can include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,organic esters such as ethyl oleate, or fixed oils including syntheticmono or di-glycerides. Aqueous solvents include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution,1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, antioxidants, chelatingagents and inert gases and the like. Those of skill in the art canreadily determine the various parameters for preparing and formulatingthe compositions of the invention without resort to undueexperimentation.

In one set of embodiments, the efficacy of various compositions of theinvention as applied to a subject may be determined or monitored bystudying the carotid arteries, which generally supply the head and neckwith blood. For example, in certain embodiments, a composition of theinvention may be applied to a subject for systemic delivery, and oneeffect of the nitric oxide may be to cause vasodilation. By studying thecarotid artery, the effect of the nitric oxide delivery may bemonitored, and if necessary, the dosing of nitric oxide adjusted. Anysuitable method may be used to monitor the carotid artery, e.g.,ultrasound, a carotid Doppler machine, functional MRI, PET scanning,etc. In other embodiments, if local delivery without systemic effects isdesired, the carotid artery can be monitored for any systemic effects.

In one aspect, one or more compositions described herein may beformulated for oral delivery. In some embodiments, one or morecompositions are provided in a capsule. A capsule can be a hard or softwater-soluble container, e.g., a gelatin container. Other examples ofcapsules include, but are not limited to polyglycolized glyceride,hydroxypropyl methylcellulose, Gelucire, iota carrageenan, hydroxypropylstarch, polyvinyl alcohol, or the like, as well as combinations of theseand/or other materials. In some cases, other materials may be added tothe capsule shell, e.g., plasticizers, coloring agents, opacifiers, orthe like. Examples of plasticizers include glycerin or sorbitol. Acapsule can be coated to affect bioavailability and/or location ofrelease. A capsule can be used to target release to gastric, duodenal,intestinal, or colonic locations within the gastro-intestinal tract of asubject. Upon release a composition can adhere to the gastric mucosa anddelivery nitric oxide to the underlying tissue. In some embodiments, acomposition described herein is mixed in a capsule with one or morefillers, diluents, glidants, or other agents. In some embodiments, acapsule wall is produced or coated to minimize oxygen and/or moisturepenetration. In some embodiments, a composition is added to a capsuleunder low oxygen and/or low humidity conditions. In some embodiments, acapsule is stored under low oxygen and/or low humidity conditions. Othercapsule materials may be found in, e.g. Challenges and Opportunities inThe Encapsulation of Liquid and Semi-Solid Formulations into Capsulesfor Oral Administration, Adv. Drug Deliv. Rev., 2008 Mar. 17;60(6):747-756, incorporated by reference herein in its entirety.

In another aspect, the present invention is directed to a kit includingone or more of the compositions discussed herein. A “kit,” as usedherein, typically defines a package or an assembly including one or moreof the compositions of the invention, and/or other compositionsassociated with the invention, for example, as described herein. Each ofthe compositions of the kit may be provided in liquid form (e.g., insolution), or in solid form (e.g., a dried powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species, which may or may not be provided with the kit.Examples of other compositions or components associated with theinvention include, but are not limited to, solvents, surfactants,diluents, salts, buffers, chelating agents, fillers, antioxidants,binding agents, bulking agents, preservatives, drying agents,antimicrobials, needles, syringes, packaging materials, tubes, bottles,flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches,containers, and the like, for example, for using, administering,modifying, assembling, storing, packaging, preparing, mixing, diluting,and/or preserving the compositions components for a particular use, forexample, to a sample and/or a subject.

A kit of the invention may, in some cases, include instructions in anyform that are provided in connection with the compositions of theinvention in such a manner that one of ordinary skill in the art wouldrecognize that the instructions are to be associated with thecompositions of the invention. For instance, the instructions mayinclude instructions for the use, modification, mixing, diluting,preserving, administering, assembly, storage, packaging, and/orpreparation of the composition and/or other compositions associated withthe kit. In some cases, the instructions may also include instructionsfor the delivery and/or administration of the compositions, for example,for a particular use, e.g., to a sample and/or a subject. Theinstructions may be provided in any form recognizable by one of ordinaryskill in the art as a suitable vehicle for containing such instructions,for example, written or published, verbal, audible (e.g., telephonic),digital, optical, visual (e.g., videotape, DVD, etc.) or electroniccommunications (including Internet or web-based communications),provided in any manner.

International Patent Application No. PCT/US2012/000151, filed Mar. 17,2012, entitled “Topical Nitric Oxide Systems and Methods of UseThereof,” by Nicholas V. Perricone, et al., is incorporated herein byreference in its entirety. In addition, the following applications, eachfiled on Sep. 19, 2012, each by Nicholas V. Perricone, are herebyincorporated by reference in their entireties: “Systems and Methods forTreatment of Acne Vulgaris and Other Conditions with a Topical NitricOxide Delivery System” (U.S. patent application Ser. No. 13/623,008);“Treatment of Skin and Soft Tissue Infection with Nitric Oxide” (U.S.patent application Ser. No. 13/623,010); “Methods and Systems forTreatment of Inflammatory Dermatoses with Nitric Oxide” (U.S. patentapplication Ser. No. 13/623,014); “Prevention and Treatment ofCardiovascular Diseases using Systems and Methods for Transdermal NitricOxide Delivery” (U.S. patent application Ser. No. 13/623,018);“Treatment and Prevention of Learning and Memory Disorders” (U.S. patentapplication Ser. No. 13/623,022); “Methods and Compositions for Muscularor Neuromuscular Diseases” (U.S. patent application Ser. No.13/622,998); “Compositions and Methods for Treatment of Osteoporosis andOther Indications” (U.S. patent application Ser. No. 13/623,004);“Techniques and Systems for Treatment of Neuropathic Pain and OtherIndications” (U.S. patent application Ser. No. 13/623,027); and “CancerTreatments and Compositions for Use Thereof” (U.S. patent applicationSer. No. 13/622,989).

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

This example illustrates one technique for preparing a composition inaccordance with one embodiment of the invention. An accurate amount of acarrier (HNC 167-62) (see below) was introduced into a system. Thecarrier weight used in these experiments was approximately 250 g and thevessel size was 500 ml. The vessel was equipped with a mechanicalstirrer, gas inlet, and gas outlet and was previously purged with argonfor about an hour. The temperature of the carrier was kept at about25-30° C. NO gas regulated at 5 psi (1 psi is about 6,900 Pa) and wasthen introduced at a controlled rate of about 1 bubble/s with continuousstiffing. The color, consistency, and viscosity of the carrier did notappear to change if NO was bubbled for 30 minutes to 2 hours. After 6hours, the weight of the carrier had increased by 0.15%, by 12 hours by0.25%, and by 24 hours by 0.56%. These increases in weight were believedto be significant considering the relative small molecular weight of NOversus the carrier. Although there was a slight change in color duringthe experiment (the color changed to slightly more orange), IR spectrumanalysis of the final product did not show any change versus the initialcarrier, indicating no noticeable chemical change in the carrier. Thecarrier also can solidify upon cooling if the carrier is initially asolid at lower temperature. Accordingly, this example demonstrates thata composition containing NO can be prepared in accordance with oneembodiment of the invention.

Example 2

In this example, six experiments were carried out to investigate theinteraction of nitric oxide with three carriers (HNC 157-62, HNC 157-65,and HNC 157-69) as well as with 1,3-propanediol, using experimentalconditions similar to that described for Example 1. In addition threeexperiments were performed to prepare carriers containing 800 ppm and500 ppm nitric oxide. HNC 157-62 was formed of 65% Phospholipon-90G(American Lecithin Company), 18% isopropyl palmitate (Kraft Chemicals),8% capric caprylic triglycerides (RITA Corp.), and 9% propanediol(Dupont). HNC 157-65 was formed of 65% Phospholipon-90G, 13% isopropylpalmitate, 14% capric caprylic triglycerides, 3% propanediol, and 5%dimethyl isosorbide (Croda). HNC 157-69 was formed from 65%Phospholipon-90G, 16% isopropyl palmitate, and 19% capric caprylictriglycerides.

The compositions were generally prepared as follows. Isopropylpalmitate, capric caprylic triglyceride, propanediol (for HNC 157-62 andHNC 157-65), and dimethyl isosorbide (for HNC 157-65) were mixedtogether and warmed to 40° C. Phospholipon-90G was then gradually addedto this liquid mixture by mixing it. Phospholipon-90G is typicallyreceived as individual pellets, and is mixed into the solution untilfully dissolved. The mixture was subsequently filtered through a sieveto remove any undissolved Phospholipon-90G.

Accordingly, the HNC carriers included 1,3-propanediol,Phospholipon-90G, isopropyl palmitate, capric and/or caporictriglycerides, and Arlasolve DMI (ICI America or Croda). Isopropylpalmitate, the capric and/or caporic triglycerides, and Arlasolve DMIare expected to be chemically inert towards nitric oxide, while theliterature suggests that 1,2-propanediol and glycerol may be able toreact with nitric oxide gas to form mononitrates. Accordingly, it wouldbe expected that 1,3-propanediol may also react with NO to formmononitrates:

In addition, Phospholipon-90G is derived from soybean and containsesters of unsaturated fatty acids such as oleic, linoleic, and linolenicacids, and thus, the unsaturated fatty acid part of Phospholipon-90Gwould react with nitric oxide to lead to a variety of nitrated products.

Each carrier was taken in a 500 mL three necked flask equipped with amechanical stirrer, gas inlet and a gas outlet. The system was purgedwith argon for one hour at room temperature (25° C.). Then nitric oxidegas was bubbled into the system. Then, nitric oxide gas was bubbledthrough carrier for stipulated amount of time. The changes in weight andcolor were noted. The details of individual experiments were as follows.

Experiment 1. The carrier was HNC 157-62. Nitric oxide gas was bubbledfor 24 hours at 25° C. The initial weight of carrier was 168.53 g., andthe final weight was 169.48 g. The net weight gained was 0.95 g and thepercentage weight gain was 0.56%.

Experiment 2. The carrier used was HNC 157-62. Nitric oxide gas wasbubbled for 48 hours at 25° C. The initial weight of carrier was 171.31g., and the final weight was 174.21 g. The net weight gained was 2.90 Gand the percentage weight gain was 1.69%.

Experiment 3. In order to differentiate between chemical reaction vs.physical absorption, the above reaction mixtures were heated at 55-60°C. for four hours. Minimal loss of weight was observed (˜200 mg),indicating no loss of absorbed nitric oxide gas. However, more intenseorange color developed during this process, indicating somedecomposition of the nitrites formed.

Experiment 4. The carrier used was HNC 157-65. Nitric oxide gas wasbubbled for 24 hours at 25° C. The initial weight of carrier was 171.66g., and the final weight was 172.98 g. The net weight gained was 1.32 gand percentage weight gain was 0.77%.

Experiment 5. The carrier used was HNC 157-69 (same as HNC 157-62,except it had no 1,3-propanediol). Nitric oxide gas was bubbled for 40hours at 25° C. The initial weight of carrier was 171.02 g., and thefinal weight was 171.97 g. The net weight gained was 0.95 g and thepercentage weight gain was 0.56%.

Experiment 6. Nitric oxide gas was bubbled through 1,3-propanediol(neat) for 40 hours at 25° C. The initial weight of the 1,3-propanediolwas 178.81 g., and the final weight was 178.97 g. The net weight gainedwas 0.16 g and the percentage weight gain was 0.09%. Experiment 7. Forpreparation of 800 ppm NO, the carrier used was HNC 157-62. Nitric oxidegas was bubbled for 2 hours at 25° C. The initial weight of carrier was238.16 g., and the final weight was 238.35 g. The net weight gained was0.19 g and the percentage weight gain 0.0798% (˜800 ppm). See entry 5 inTable 1.

Experiment 8. For preparation of 500 ppm NO, the carrier used was HNC157-65. Nitric oxide gas was bubbled for 2 hours at 25° C. The initialweight of carrier was 250.37 g., and the final weight was 250.50 g. Thenet weight gained was 0.13 g and the percentage weight gain was 0.0519%(˜500 ppm). See entry 6 in Table 1.

Experiment 9. For preparation of 800 ppm NO, the carrier used was HNC157-62. Nitric oxide gas was bubbled for 15 min at 25° C. The initialweight of carrier was 252.24 g., and the final weight was 252.45 g. Thenet weight gained was 0.21 g and the percentage weight gain 0.083% (˜800ppm).

These experiments were conducted with carriers the HNC 157-62, HNC157-65, HNC 157-69, and 1,3-propanediol.

As described above and in Table 1, weight gains ranging from 0.5% to1.7% were observed when nitric oxide gas was passed through thecarriers. In order to determine the nature of interaction between nitricoxide and carrier, the carrier was heated after nitric oxide absorptionat 60° C. for four hours. Practically no loss of weight was observed,which indicated that the nitric oxide gas reacted chemically with thecarriers (entries 1-4 in Table 1).

In order to investigate the reactivity of 1,3-propanediol with nitricoxide, nitric oxide absorption was studied using (a) HNC 157-69, whichdid not contain 1,3-propanediol, and (b) 1,3-propanediol by itself. HNC157-69 gained 0.95 g or 0.56% weight, much lower compared to its1,3-propanediol containing analog HNC 157-62, which showed 1.69% weightgain (entries 2 and 5 of Table 1). 1,3-propanediol itself, surprisingly,showed only negligible, if any, weight gain when NO was passed throughit (entry 6 in Table 1). Thus, under experimental conditions,1,3-propanediol did not react with nitric oxide.

Two samples were also prepared containing 800 ppm NO (from carrier HNC157-62) and one sample containing 500 ppm NO (from carrier HNC 157-65)(entries 7-9 in Table 1). The IR spectra of the carriers did not showany additional bands after the reaction, possibly because of low amountsof nitrites and/or overlap with the carrier complex bands.

Mass spectral studies of the carrier HNC 157-62 and HNC 157-62containing NO indicated that there was an increase in the intensity ofthe peak at m/e 104 in NO-containing carrier, compared to carrierwithout NO. The peak at m/e 104 was believed to be due to choline cation(C₅H₁₄NO). Phospholipon-90G may contain some free choline, and hencepresence of the peak at 104 in the mass spectrum of the carrier was notsurprising. However, the increase in the amount of choline after passageof NO was somewhat unexpected, although it is believed that nitric oxidecatalyzes similar dephosphorylation of Phospholipon-90G releasingcholine.

In conclusion, an increase in weight (0.56 to 1.69%) was observed whennitric oxide gas was passed through the carriers. 1,3-propanediol failedto gain any significant weight when nitric oxide was passed through it.HNC 157-69 (devoid of 1,3-propanediol) gained only 0.56% weight comparedto 1.69% by its 1,3-propanediol containing analog HNC 157-62. The massspectra of HNC 157-62 before and after passing NO indicated that thepeak corresponding to choline at m/e 104 increased after the passage ofNO, which suggests that phospholipon-90G may undergo NO-catalyzeddephosphorylation.

TABLE 1 Wt. % Expt. Initial Final Time Temp Gain Wt. No. Carrier wt. g.wt. g. hr ° C. g gain 1 HNC 157-62 168.53 169.48 24 25 0.95 0.56 2 HNC157-62 171.31 174.21 48 25 2.90 1.69 3 HNC 157-62 174.21* 174.01 4 60−0.20 −0.11  4 HNC 157-65 171.66 172.98 24 25 1.32 0.77 5 HNC 157-69171.02 171.97 40 25 0.95 0.56 6 1,3- 178.81 178.97 40 25 0.16 0.09Propanediol 7 HNC 157-62 238.16 238.35 2 25 0.19 0.0798 (~800 ppm) 8 HNC157-65 250.37 250.50 2 25 0.13 0.0519 (~500 ppm) 9 HNC 157-62 252.24252.45 0.25 25 0.21 0.0833 (~800 ppm)

Example 3

This example illustrates non-invasive blood pressure measurements inmice using a composition in accordance with one embodiment of theinvention.

Blood pressure in mice may be measured using blood volume changes in themouse tail. Mice with normal tails (no clipping or short) were used inthis study. Ages varied between 8 weeks and 24 weeks. This procedureuses the CODA non-invasive blood pressure system available from KentScientific (Torrington, Conn.) Mice weighing approximately 25 grams wererestrained in plastic cylindrical housing with a nose come allowing thenose to protrude. Two tail cuffs provided occlusion and measurements.The O-cuff provided period occlusion while the VPR cuff providesvolume-pressure recordings. The occlusion pressure and the recordedpressure were controlled automatically by the computer software.

Each measurement had 10 acclimation cycles and 20 measurement cyclesonce daily depending on the experimental parameters. The average bloodpressure of male mice made over time was 136/88. In this study, bloodpressure measurements of the control (base) mice and the test (treated)mice were made and averaged over a period of three days. The testcomposition comprised 800 ppm nitric oxide, which was applied over theupper back of the animal in a quantity that exceeded 50 mg. The bloodpressures were recorded after each application and over 1 hour. Thefollowing results were obtained.

TABLE 2 Average Blood Pressure Reading Post Application Control (Base)Product Test (Treated) NO 800 ppm Time 0 167/123 140/91 1 Hour 170/128115/69This study shows that the application of the test product is capable ofreducing the blood pressure in a mouse to a significantly lower value.

Example 4

This example illustrates capillary blood flow measurements in humansusing a composition similar to the ones described in the above examples.

Microcirculation properties of the skin were measured before and afterapplication of a test product with nitric oxide. Measurements were madeusing a Moor® Laser Doppler instrument. Measurements were made at Day 1before and immediately, 5 and 15 minutes after treatment.

The study participants were healthy females aged 30 to 55 years. Theywere in good health as determined by the medical history and were nottaking any prescription medications. The test product was labeled as 800ppm NO (nitric oxide). The study participants had 100 mg of the testproduct applied in a 2×2 sq. inch area on the forearm.

Laser Doppler was performed to measure increased stimulation of themicro-capillary blood flow to skin. The micro-circulation of the skinreflects the perfusion of the skin and the underlying tissue. The laserDoppler technique is the standard method to obtain dynamic measurementof capillary blood flow in clinical evaluation. Measurements can be maderelatively rapidly and simultaneously at sites. In addition, temperaturemeasurements may also be made at the same time.

A Moor Instruments DRT4 Laser Doppler Blood Flow Monitor (Devon,England) was used. The laser Doppler technique measures blood flow inthe microcapillaries of the skin that are close to the skin surface andthe blood flow in underlying arterioles and venules that help toregulate skin temperature. There are several parameter used to describeblood flow measured by this laser Doppler technique. These measurementparameters are defined by Moor Instruments Inc. and are listed below.

Flux: This parameter is related to the product of average speed andconcentration of moving red blood cells in the tissue sample volume. Itis the parameter most widely reported in Laser Doppler publication.

Conc: This parameter gives an indication of the number of moving redblood cells in the tissue sample volume.

Speed: This parameter gives an indication of the average speed of redblood cells moving in the tissue sample volume.

Temp: This is the probe temperature and where there is good thermalconduction between probe and tissue it reaches tissue temperature.

Because of the nature of blood flow in the capillaries and other smallblood vessels, absolute flow units such as ml/minute cannot beexpressed. Therefore, arbitrary units are used. Blood flow changes aredefined as the percentage change from the baseline of these arbitraryunits.

The procedure was as follows. The laser Doppler probe was attached ontothe volar forearm. Control untreated skin readings (Baseline) wereobtained for 15 minutes. The test product was then applied in thedesignated 2×2 sq. inch area and rubbed into the skin. Readings wereobtained for 15 minutes.

The areas not used for evaluation include the first 15 seconds afterstarting data collection. Four 10 second areas in the baseline and testreadings at each time point were randomly selected to obtain the meanaverages which were then used in further analysis of the data.

The averaged data was compiled from the 4 study participants and thelaser Doppler results are provided in FIG. 1. There was a significantdifference observed in the control untreated skin and the skin treatedwith 800 ppm nitric oxide up to 15 minutes after application. Theapplied nitric oxide had an effect on the micro circulation of the skinat the applied level (100 mg in a 2×2 sq. inch area). The topical testproduct is capable of passing through the skin and affecting themicrocirculation of the skin.

Example 5

This example illustrates delivery of nitric oxide formulations similarto those discussed herein in humans. In these studies, laser Dopplerstudies were performed on three human female subjects. As discussedbelow, all of the studies showed positive results consistent with aphysiological effect of nitric oxide applied to the skin, passingthrough the skin and affecting the capillary circulation. Theformulations used produce positive, almost immediate results whenapplied to the skin which, in these studies, was manifested byvasodilatation of the cutaneous vascular system.

While nitric oxide has many physiological effects, the purpose of thesestudies was to measure physiological effects that would be relativelyeasy to determine, and which would be noninvasive. Laser Doppler wasselected for these studies because laser Doppler has a relatively largedatabase that indicates that it is effective in determining an increasein microcirculation, i.e., circulation within the capillary bedimmediately under the epidermal layer in the skin. Nitric oxide iscapable of violating the capillary bed, and thus, laser Doppler wasselected.

The formulation used in these studies contained nitric oxide dispersedin a lipid matrix. The nitric oxide was dispersed in the matrix and doesnot appear to be dissolved but remained intact, i.e., it appeared todiffuse into the skin as a molecule of nitric oxide rather than asatomic components or ions. Nitric oxide is a very rapid acting molecule,and these studies used a system that employed laser Doppler with acovered chamber. The formulation was placed into the chamber and thenattached to the skin by an adhesive layer on the covering. This provideda stable measuring device as determined by multiple normal evaluationsof the capillary blood flow without treatment of nitric oxide.

A known positive control, methyl nicotinate, was applied to the skin ata concentration of 1/10% in alcohol. There was a rapid response typicalof the vasodilator. The vascular dynamics are such that when the bloodvessels dilate physical parameters follow Bernoulli's law. This statesthat as one increases the diameter of a tube containing a liquid to flowwill increase but the pressure and the speed of the liquid willdecrease. The laser Doppler device accurately measured these parameters.

When nitric oxide was applied to the forearm of three human femalesubjects, it was observed that there was an immediate effect on theblood flow as soon as the formulation was applied. The speed of bloodflow decreased, and the effects lasted over 15 minutes. There was noerythema and no discomfort to the subjects that was observed.

Thus, these studies showed that the nitric oxide in the lipid matrixwhen applied topically to the forearm, was able to penetrate the skinvery rapidly and to interact with the underlying tissues. This isevidenced by the observation of vascular dilatation without erythema.Accordingly, it can be concluded from these studies that the formulationcontaining nitric oxide was effective in delivering nitric oxide throughthe skin in a physiologically active state.

In these studies, the participants were healthy females aged 30 to 55years. They were in good health as determined by the medical history andwere not taking any prescription medications. All study participantsread and signed the informed consent statement prior to any studyprocedures being performed.

The test product was labeled as 10,000 ppm NO (nitric oxide). The studyparticipants had 100 mg of the test product applied in a closed Hilltopchamber on the forearm.

Laser Doppler was performed to measure increased stimulation of themicro-capillary blood flow to skin. The micro-circulation of the skinreflected the perfusion of the skin and the underlying tissue. LaserDoppler is a standard method to obtain dynamic measurement of capillaryblood flow in clinical evaluation. Measurements can be made relativelyrapidly and simultaneously at sites. In addition, temperaturemeasurements may also be made at the same time.

A Moor Instruments DRT4 Laser Doppler Blood Flow Monitor (Devon,England) was used in these studies. The laser Doppler technique measuredblood flow in the microcapillaries of the skin that are close to theskin surface and the blood flow in underlying arterioles and venulesthat help to regulate skin temperature. Because of the nature of bloodflow in the capillaries and other small blood vessels, it is difficultto determine absolute flow units such as ml/minute. Therefore, arbitraryunits were used in these experiments to determine relative changes.Blood flow changes were accordingly defined as the percentage changefrom the baseline using the arbitrary units.

Data was compiled from the three study participants and the laserDoppler studies and averaged. It observed that there was a significantdifference observed in the control untreated skin and the skin treatedwith 10,000 ppm nitric oxide up to 15 minutes after application. Theapplied nitric oxide had an effect on the microcirculation of the skinat the applied level (100 mg in a lipid matrix). The formulation wasdetermined to be capable of passing through the skin and affecting themicrocirculation of the skin.

Example 6

This example illustrates a protocol used for determining the amount ofnitric oxide released from various compositions of the presentinvention. The protocol is generally performed as follows:

1) Samples (within HNC carriers described herein) were maintained ateither 4° C. or −20° C. until analyzed.

2) One at a time and before they were opened, samples were warmed to 37°C. (dry bath) in a controlled atmosphere of 1% O₂. They were opened and150 microliters was removed and placed into 1.35 mL of PBS (pH 7.4, 25°C. and equilibrated to 13 micromolar O₂) in a small glass screw-cap vialminimizing headspace.

3) The vials were then subjected to 15 s of vortex agitation, capsopened and 1 mL quickly removed and immediately injected into a SieversNitric Oxide Analyzer reaction chamber containing 4 mL of deionized anddistilled H₂O equilibrated at 0% O₂ by a N₂ flow through gas andmaintained at 37° C. by flow-through water jacket.

4) Peaks were analyzed by comparing values to a standard curve generatedby injecting various concentrations of the NO donor1-(hydroxy-NNO-azoxy)-L-proline (PROLI-NONOate). To standardize amongsamples, the area under the curve from 0 to 4 min was used forconcentration determinations are expressed in moles of NO and have beencorrected for the 2 dilutions (50×). As such, these values equal molesof NO/150 microliter matrix.

5) The authenticity of NO formation was validated by injecting 50 mM ofthe NO spin trap2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide(cPTIO) or by injecting sample into the reaction chamber containing 50mM cPTIO and observing the absence of signal.

6) For each sample, the area under the curve from t=0 to t=4 mM wascalculated for concentration determinations. Calculations represent onlythe amount of NO released during this 4 mM time span and thus do notrepresent the total amount of NO contained in the volume of matrixassessed (150 microliters). For each vial 3-5 independent determinationswere performed (this means each injection represents removal of 150microliters of matrix from the labeled vial, dilution in PBS andinjection. As such, these were completely independent measurements frombeginning to end.

TABLE 3 Sample ID Mean (millimoles NO) Std Dev 1000 11.4 1.55 4000 60.71.70 7000 72.1 11.9 10000 127.9 11.3In this table, the Sample ID numbers correspond to the amount of nitricoxide, in ppm, that was formulated in the composition (sample) tested.

Based on these results, the compositions tested here appeared to beeffective in entrapping nitric oxide gas, and are effective in releasingthe trapped gas in a measurable and significant way.

Example 7

This example illustrates delivery of nitric oxide formulations to fivehuman subjects, using procedures and formulations similar to thosediscussed in Example 5. Each person served as their own control for thetesting. A Moor Instruments Laser Doppler was used to determinecirculation. All of these studies showed positive results consistentwith a physiological effect of nitric oxide applied to the skin, passingthrough the skin and affecting the capillary circulation. Theformulations used produce positive, almost immediate results whenapplied to the skin which, in these studies, was manifested byvasodilatation of the cutaneous vascular system.

For each subject, the following creams were applied: a negative controlformulation (containing no NO), 0.010 gram of a 10,000 ppm NOformulation, 0.020 gram of a 10,000 ppm NO formulation, and a positivecontrol formulation containing 0.1% methyl nicotinate. The formulationswere prepared using procedures similar to those discussed in Example 1.

When nitric oxide was applied to the forearm of the subjects, it wasobserved that there was an immediate effect on the blood flow as soon asthe formulation was applied. The speed of blood flow decreased, and theeffects lasted over 15 minutes. There was no erythema and no discomfortto the subjects that was observed.

Data was compiled from the study participants and the laser Dopplerstudies and averaged. It observed that there was a significantdifference observed in the control untreated skin (where no detectablevasodilation was observed), and the treatments involving 0.010 g of NO,0.020 g of NO, and methyl nicotinate. For all three treatments,vasodilatation was observed, with somewhat greater vasodilatation forthe 0.010 g formulation over the 0.02 g formulation. In addition, theamount of vasodilatation observed for the 0.01 g formulation of NO wasgenerally similar to the amount of vasodilatation observed for methylnicotinate.

Accordingly, these data demonstrate that NO as applied in a cream to theskin is able to penetrate the skin and affect bloodflow beneath theskin.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method, comprising: administering, to the skinof a subject having or at risk of osteoporosis, a composition comprisingan effective amount of molecular nitric oxide and a carrier comprisingphosphatidylcholine entrapping the molecular nitric oxide, and a secondphase comprising an emulsifier, wherein the molecular nitric oxide ispresent within the carrier as a gas or bound by hydrogen bonds or vander Waals forces, wherein the composition is stable at room temperature.2. The method of claim 1, wherein the phosphatidylcholine carrierstabilizes the nitric oxide at a temperature at or lower than 80° C. 3.The method of claim 1, wherein the composition comprises a liquidcrystal structure comprising the phosphatidylcholine.
 4. The method ofclaim 3, wherein at least a portion of the liquid crystal structure ismultilamellar.
 5. A method, comprising contacting the skin of a subjecthaving or at risk of osteoporosis, with a composition comprising: anemulsion comprising a first phase comprising molecular nitric oxide andlecithin, and a second phase comprising an emulsifier, wherein thelecithin is present at least about 0.25% by weight of the composition,wherein the first phase comprises no more than about 250 ppm of water byweight of the composition, and wherein the molecular nitric oxide ispresent within the emulsion as a gas or bound by hydrogen bonds or vander Waals forces to the lecithin.
 6. The method of claim 5, wherein thefirst phase forms discrete vesicles contained within the second phase.7. The method of claim 6, wherein the first phase forms liposomescontained within the second phase.
 8. The method of claim 7, wherein thefirst phase forms multilamellar liposomes contained within the secondphase.
 9. The method of claim 5, wherein the nitric oxide is present atat least about 0.5% by weight of the composition without nitric oxide.10. The method of claim 5, wherein the lecithin comprises aphosphatidylcholine.
 11. The method of claim 10, wherein at least someof the phosphatidylcholine comprises a phosphatidylcholine comprising astearic diglyceride linked to a choline ester of a phosphoric acid. 12.The method of claim 10, wherein at least some of the phosphatidylcholinecomprises a polyenylphosphatidylcholine.
 13. The method of claim 12,wherein the polyenylphosphatidylcholine comprises linoleic acid.
 14. Themethod of claim 5, wherein the first phase further comprises atransdermal penetration enhancer.
 15. The method of claim 5, wherein thefirst phase comprises a fatty acid ester.
 16. The method of claim 5,wherein the first phase comprises ascorbate palmitate.
 17. The method ofclaim 5, wherein the first phase comprises isopropyl palmitate.
 18. Themethod of claim 5, wherein the composition is a gel.
 19. The method ofclaim 5, wherein the composition is a cream.
 20. The method of claim 5,wherein the composition is substantially transparent.